Enzyme formulation and method for degradation

ABSTRACT

An enzyme formulation includes an encapsulated fungal enzyme which is effective for degrading at least one material selected from the group consisting of hydrocarbons, vulcanized rubber, synthetic rubber, natural rubber, vulcanized polymers and perfluorinated compounds. A degradation method includes treating one of the above-mentioned materials with an encapsulated fungal enzyme to degrade the material.

BACKGROUND OF THE INVENTION

Hydrocarbon contamination resulting from drilling and extraction of oil has become one of the major environmental problems. Accidental releases of petroleum products are of particular concern for human health since hydrocarbon components cause extensive damage to the environment and contaminate the soil. The microbially mediated breakdown of heavy weathered total petroleum hydrocarbons (TPH) has its limitations due to the degradation of only up to 4-ring aromatic compounds and 25-carbon saturated compounds. Moreover, the presence of polycyclic aromatic hydrocarbons (PAHs) with two or more fused benzene rings in linear, angular or cluster structural arrangements and low solubility poses an additional remediation challenge.

Therefore, it would be desirable to provide a new enzyme formulation and method for enhanced degradation of hydrocarbons. The enzyme formulation can be particularly useful for enhanced remediation of hydrocarbon contaminated soil matrices.

It would also be desirable to provide an enzyme formulation and method for the degradation of vulcanized rubber, synthetic rubber, natural rubber, vulcanized polymers and perfluorinated compounds.

SUMMARY OF THE INVENTION

An enzyme formulation comprises an encapsulated fungal enzyme which is effective for degrading at least one material selected from the group consisting of hydrocarbons, vulcanized rubber, synthetic rubber, natural rubber, vulcanized polymers and perfluorinated compounds.

A degradation method comprises treating one of the above-mentioned materials with an encapsulated fungal enzyme to degrade the material.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The ability of ligninolytic enzymes to degrade larger molecular compounds through the use of radical reactions brings potential to enhance the degradation of crude oil and other hydrocarbons. Since ligninolytic enzymes are extracellular, they are able to diffuse effectively to the highly immobile high molecular weight hydrocarbons and result in metabolites that are more bioavailable for further microbially induced breakdown.

The present invention includes ligninolytic enzymes which are encapsulated to stabilize the enzymes. The invention can further include formulating the stabilized enzymes for a specified application such as for the breakdown of TPHs.

The invention can further include the encapsulation of other species of fungal enzymes for use in breaking down other materials.

Example of Process for Producing Ligninolytic Enzymes:

Fungal species of, but not limited to, Phanaerochete chrysosporium, Nematoloma forwardii or Trametes versicolor are cultivated in flask and bioreactor cultures in standard conditions. Other examples of fungal species and known producers of ligninolytic enzymes include Phanerachete spp, Tremetes spp, Phlebia spp, Cerena spp, Merulius spp, Pellinius spp, Cyatus spp, and Stereum spp. The selected fungus secretes extracellularly into the growth medium a suite of ligninolytic enzymes: manganese peroxidase, lignin peroxidase and laccase. The enzyme activity can be analyzed using standard methods.

The use of purified enzyme results in the highest possible activity. Once the activities of manganese peroxidase and lignin peroxidase reach maximum, the extracellular liquids with growth medium are collected. The fungal mycelium is separated from the liquid components via sterile gauze and incinerated. The remainder of extracellular liquids is then filtered and purified using standard methods. Purified enzyme suite is then lyophilized and utilized for further stabilization.

Any suitable ligninolytic enzyme(s) can be used in the invention. The major groups of ligninolytic enzymes include lignin peroxidases, manganese peroxidases, versatile peroxidases, and laccases, examples of which are known to enzyme scientists.

Although the enzymes may be a suite of enzymes produced by a fungus, alternatively a single type of ligninolytic enzyme may be used. Further, the enzyme(s) are not necessarily produced by a fungus, but could instead be produced by a microorganism or other source. Although purified enzymes are preferred, in certain embodiments the enzymes may be used in nonpurified form.

Enzymes Stabilization:

The enzymes are stabilized by encapsulation. For example, the enzymes can be encapsulated in a protective shell. In certain embodiments, the type of encapsulation is microencapsulation. The shell can be any material that is effective to stabilize the enzymes. In certain embodiments, the shell is effective to cause slow release of the enzymes. For example, in one enzyme release experiment the encapsulated enzymes had a very low enzyme activity the first 7 days, and the enzyme activity gradually increased to a high activity between days 7 and 28.

In certain embodiments, the shell consists of a cross-linked hydrogel. Crosslinking is a way of curing the hydrogel. The process of crosslinking initiates from the outer layer and progresses to the core; in this way the enzymes are forced to stay inside the hydrogel. Furthermore, the crosslinking reaction provides rigidity to the hydrogel. It is also believed that a crosslinker such as manganese will get involved into the enzyme catalytic cycle and replenish manganese as needed during the decontamination process.

The cross-linked hydrogel is exemplified by but not limited to calcium alginate, manganese alginate, zirconium alginate, calcium poly(aspartate), manganese poly(aspartate) and zirconium poly (aspartate).

More generally, a good match of the encapsulating (shell) material and the enzymes may be determined based on the concentration, and the valency of the cation. For example, higher concentration and higher valent cation (Zr+4 is more effective than Ca+2) increases the gelation time.

In another embodiment the enzymes are stabilized by encapsulation in a shell having two or more layers. For example, the protective shell may comprise two layers wherein the first layer interfaced with the enzyme is a cross-linked hydrogel, and the second layer interfaced with the first layer is a hydrophobic material such as an oleogel. The two layer shell is for swelling the contaminants in the outermost layer followed by oxidation reaction in the inner layer when they come in contact with the enzyme. The hydrophobic material can attract and be attracted to hydrocarbons.

The oleogel is exemplified by but not limited to poly(lauryl methacrylate), poly(stearyl methacrylate), poly(isoprene) and poly (butadiene).

In certain embodiments, the encapsulant has one or more of the following benefits: room temperature process, bio-based and biodegradable matrix, absorbs water, fast synthesis, VOC free/no solvents, variable particle size, and stable pH 4 to 6 range.

In certain embodiments, the shell works as a donor of one or more mediators for activation of enzymes within the capsule. For example, as described above, a manganese crosslinker can replenish manganese to activate the enzyme catalytic cycle. In certain embodiments, the shell is formulated to attract oil molecules or other material to be degraded.

The encapsulated enzymes can be produced in the form of beads or any other form suitable for a particular application. In certain embodiments, the beads have a diameter within a range of from about 1.5 mm to about 5 mm, and more particularly within a range of from about 2.8 mm to about 3.5 mm (“diameter” refers to maximum diameter). In several examples, ligninolytic enzymes have been encapsulated in an alginate shell to produce spherical beads having diameters of 1.9 mm, 2.8 mm and 3.0 mm.

Example

The process of stabilizing the enzyme is further exemplified in the following example. 10 mg of manganese peroxidase was rinsed into 10 g of alginate stock solution with 1 mL of deionized water. The suspension was mixed until dissolve with vial mixer and uniform. 10 grams of alginate mixture was then drawn up into a 10 mL syringe. Alginate drops containing manganese peroxidase were dropped using 0.3 mm gauge needle into the 50 mL calcium chloride solution using syringe pump (Cole-Palmer 78-0100C) with the retention time of 100 mL/h. Formation of encapsulated gel-like beads of alginate-enzyme complex was detected. The enzyme capsules were then left to settle at the bottom of the container and refrigerated at 8 C until use.

Formulating the Stabilized Enzyme for a Specified Application:

The invention further includes formulating the stabilized enzyme for specified application. In one example, the stabilized enzyme is formulated in a liquid or solid matrix. The matrix comprises a peroxide such as hydrogen peroxide or its derivatives and dispersing aid such as surfactants.

Possible Market and Product Applications:

Encapsulated fungal enzymes in the environmental setting for the purpose of degradation of total petroleum hydrocarbons may be of interest to oil and gas companies. Furthermore, since radical reaction pathway of fungal ligninolytic enzymes is highly unspecific, the application of the technology may be broad ranging from: degradation of heavily weathered petroleum hydrocarbons, petroleum hydrocarbons, jet fuel, Navy special fuel, polyfluorinated compounds (PFCs), dioxins; PCBs, herbicides, pesticides, munition constituents, lubricants, oils, detoxification of industrial effluents, and dye effluents. The enzyme formulation and method can be useful for the degradation of vulcanized rubber, synthetic rubber, natural rubber, vulcanized polymers and perfluorinated compounds such as perfluorooctanesulfonic acid and perfluorooctane sulfonate.

Evaluation of Fungal Enzyme Extracts to Catalyze Remediation of Heavily Weather Crude Oil Contaminated Soil:

Objective: To develop a method to treat heavily weathered crude contaminated soil to <1% TPH encapsulated fungal enzymes.

Hypothesis: Fungal enzymes can non-selectively break down long-chain hydrocarbons into shorter chain hydrocarbons that can be further degraded by microorganisms.

Phanerochaete chrysosporium

Secrete a suite of oxidoreductases (manganese peroxidase, laccase and lignin peroxidase).

The cation radical of heme porphyrin reacts with an array of compounds and initiates non specific of recalcitrant environmental pollutants. (See FIG. 2)

Enzyme Characteristics:

Non specific degradation of recalcitrant environmental pollutants e.g. TPH.

Dosage for treating recalcitrant contaminants—1 U/1.89 mg/kg PAH.

Encapsulation:

To provide reactive ingredient (enzyme) in an easily applicable form without the risk of introducing non native fungal species.

Examples of Encapsulation Technology:

Microencapsulation via spray drying with mixture of polymer and solvent (solid material)

Encapsulation into hydrogel particles using non aqueous dispersion process

Encapsulation using complex co-aservation

Encapsulation via electrospray

Selection of Encapsulant:

Match suitable encapsulation route with critical process metrics to gain high probability of success.

Ionotropic Alginate Gellation Benefits:

Room temperature process

Bio-based and biodegradable matrix

100% aqueous

Fast process

VOC free/No solvents

Variable particle size

Contaminated Samples:

Grand Calumet River Sediments

Contamination from multiple industries including oil refineries on the banks of the river

Contamination in place since 1970's

Contaminants include PCBs, heavy metals, crude oil, PAHs, heavily weathered petroleum hydrocarbons, petroleum hydrocarbons, vulcanized rubber, jet fuel, Navy special fuel, synthetic rubber, polyfluorinated compounds (PFCs), dioxanes; PCBs, herbicides, pesticides, and munition constituents.

Soil Characteristics Concentration (mg/kg dry weight) TPH in Soil  C6-C12 ND >C12-C28 1,530 >C28-C35 581 Total C6-C35 2,110 PAHs in Soil Acenaphthene 0.231 Acenaphthylene 1.45 Benzo[a]anthracene 9.78 Chrysene 31.6 Phenanthrene 19.1 Fluoranthene 15.31 Soil Moisture Content 17%

Metagenomics and Metaproteomics:

To understand the shift in microbial population as a result of application of fungal enzymes and degradation of TPH

To detect suite of microbial proteins directly involved in TPH degradation

Use data to optimize treatment

Application of Omic Technologies:

Biodegradation: Baseline and time/dose response characterization

Community structure (microbes)

Functional potential (genes)

Function (proteins)

Additional Work:

Optimize encapsulation conditions

Conduct experiment with encapsulated enzyme formulation

Analysis of metaproteome after treatment with encapsulated enzyme to compare protein composition

Application to Field Treatment:

Formulation of encapsulated enzyme with hydrogen peroxide embedded

Apply encapsulated enzyme into vadose zone soils using backhoe

Encapsulant is resistant to mechanical stress due to size

Reaction is expected to occur rapidly with the reduction in TPH seen within 28-30 days after application

Measure TPH concentration to determine when to reapply enzyme

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the experimental setup for alginate encapsulation of enzyme.

FIG. 2 illustrates the reaction pathways of heme porphyrin with various reactants.

FIG. 3 illustrates the molecular structure of a tetrasaccharide monomer of the ionotropic alginate ion polymer.

EXPERIMENTATION Laboratory Experiment Approach: Dry Soil

Grow culture of P. chrysosporium and collect extracellular enzyme extract Lyophilize enzyme extract and resuspend in 10 ML Apply to soil microcosm and add hydrogen peroxide (reaction substrate)

pH 4.5, Temp 25 C

Treatments Set Up Soil Enzyme Peroxide 1 ✓ 2 ✓ ✓ 3 ✓ ✓ ✓ 20 g soil 2 mL purified enzyme at 20 U/mL 100 μL 10 mM hydrogen peroxide added every other day TPH and PAH measured after 7 days and 14 days All treatments prepared in duplicates

Results - Soil, Enzymes + H₂O₂, Day 7 Day 0 Day 7 Percent (mg/kg dry weight) (mg/kg dry weight) Loss TPH in Soil  C6-C12 ND >C12-C28 1,530 >C28-C35 581 Total C6-C35 2,110 PAHs in Soil Acenaphthene 0.231 Acenaphthylene 1.45 Benzo[a]anthracene 9.78 Chrysene 31.6 Phenanthrene 19.1 Fluoranthene 15.31

ADDITIONAL EXPERIMENTS 1. Selection of Fungal Strains for Production of High Concentrations of Enzymes: Manganese Peroxidase and Laccase.

Exp. 1. Phanerachaete chrysosporium 1309, Lenzites betulina 141, Trametes versicolor 159, Trametes cervina 33, Trametes ochraceae 1009, Trametes pubescens 11, Stereum hirsutum 42, Trametes zonatus 540, Trametes hirsuta 119, Phlebia radiata 312. Composition of synthetic medium, (g/l): KH₂PO₄—1.0; MgSO₄—0.5; CaCl₂—0.1; FeSO₄×7H₂O—0.005; peptone—2.0; yeast extract—2.0; glycerol—10.0; veratryl alcohol—0.3, pH 5.0.

Laccase, U 1⁻¹ pH Cultivation days 4 6 8 11 4 6 8 11 P. chrysosporium 1309 0 0 0 0 5.0 6.4 6.5 6.5 L. betulina 141 0 0 0 0 6.0 6.5 7.1 7.0 T. versicolor 159 2352 890 613 0 6.2 7.3 6.7 6.4 T. cervina 33 0 0 0 0 6.2 5.8 5.6 6.7 T. ochraceae 1009 121 252 231 111 6.0 5.9 6.1 6.0 T. pubescens 11 0 8 3 0 5.9 5.0 5.7 6.0 S. hirsutum 42 3 0 0 0 5.0 5.0 5.0 5.0 T. zonatus 540 4200 3276 2394 2100 5.8 6.2 6.8 6.6 T. hirsuta 119 256 143 336 806 4.8 4.3 6.0 5.5 P. radiata 312 17 8 10 15 6.1 5.8 5.8 5.3 MnP, U 1⁻¹ (610 nm) MnP, U 1⁻¹ (270 nm) Cultivation days 4 6 8 11 4 6 8 11 P. chrysosporium 1309 0 0 0 0 0 103 43 0 L. betulina 141 0 0 0 0 0 0 0 9 T. versicolor 159 77 890 16 0 99 60 21 0 T. cervina 33 59 0 559 254 21 125 168 236 T. ochraceae 1009 48 252 205 87 176 280 267 232 T. pubescens 11 0 8 0 0 0 0 0 0 S. hirsutum 42 0 0 0 0 0 9 9 13 T. zonatus 540 86 3276 144 100 146 112 99 86 T. hirsuta 119 0 143 0 0 13 9 9 17 P. radiata 312 0 8 0 0 0 9 0 0 LiP, U 1⁻¹ Cultivation days 4 6 8 11 P. chrysosporium 1309 2 6 3 3 L. betulina 141 1 2 2 4 T. versicolor 159 9 4 2 2 T. cervina 33 1 1 3 6 T. ochraceae 1009 4 6 19 19 T. pubescens 11 2 1 8 2 S. hirsutum 42 0 0 1 1 T. zonatus 540 2 7 14 25 T. hirsuta 119 6 5 1 3 P. radiata 312 9 13 5 8 Composition of medium, (g/l): KH₂PO₄—1.0; MgSO₄—0.5; CaCl₂—0.1; FeSO₄×7H₂O—0.005; peptone—1.0; yeast extract—2.0; veratryl alcohol—0.3; MP—40.0. pH 5.0.

Laccase, U 1⁻¹ pH Cultivation days 4 6 8 11 4 6 8 11 P. chrysosporium 1309 0 0 0 0 4.2 5.8 6.8 7.4 L. betulina 141 0 0 0 0 5.8 6.0 6.4 6.5 T. versicolor 159 106 143 235 0 5.1 6.2 6.1 6.0 T. cervina 33 0 0 0 0 5.2 5.4 6.2 6.3 T. ochraceae 1009 5544 5432 1596 722 3.2 5.0 5.2 5.6 T. pubescens 11 0 0 0 0 5.3 5.0 5.4 4.7 S. hirsutum 42 168 223 164 69 4.0 4.9 4.7 5.0 T. zonatus 540 8400 7896 5796 2520 3.8 5.2 5.2 5.7 T. hirsuta 119 1276 353 67 22 4.0 4.6 4.3 4.0 P. radiata 312 0 0 0 0 5.0 5.1 5.6 4.2 MnP, U 1⁻¹ (610 nm) MnP, U 1⁻¹ (270 nm) Cultivation days 4 6 8 11 4 6 8 11 P. chrysosporium 1309 0 0 0 0 0 0 0 0 L. betulina 141 0 0 0 0 17 0 0 13 T. versicolor 159 0 0 0 0 9 9 0 9 T. cervina 33 46 966 846 742 0 615 512 396 T. ochraceae 1009 100 164 171 104 374 318 310 387 T. pubescens 11 0 0 0 0 0 0 0 0 S. hirsutum 42 0 0 0 0 17 17 0 0 T. zonatus 540 129 103 97 99 129 215 159 172 T. hirsuta 119 0 0 0 0 30 9 26 0 P. radiata 312 0 0 0 0 0 0 0 4 LiP, U 1⁻¹ Cultivation days 4 6 8 11 P. chrysosporium 1309 192 0 7 0 L. betulina 141 0 0 0 11 T. versicolor 159 0 15 37 15 T. cervina 33 0 0 0 0 T. ochraceae 1009 24 8 14 76 T. pubescens 11 0 0 0 3 S. hirsutum 42 0 2 1 38 T. zonatus 540 27 16 20 55 T. hirsuta 119 21 18 14 22 P. radiata 312 0 0 0 11 Exp. 2. Cerrena unicolor 300, Cerrena unicolor 301, Cerrena unicolor 302, Cerrena unicolor 303, Cerrena unicolor 305, Coriolopsis gallica 142, Merulius tremelosus 206, Pellinus tuberculosus 121, Pellinus tuberculosus 131, Cyatus striatus 978. Composition of synthetic medium, (g/l): KH₂PO₄—1.0; MgSO₄—0.5; CaCl₂—0.1; FeSO₄×7H₂O—0.005; peptone—2.0; yeast extract—2.0; glycerol—10.0; veratrylalcohol—0.3, pH 5.0.

Laccase, U 1⁻¹ pH Cultivation days 5 7 9 12 5 7 9 12 C. unicolor 300 336 468 286 798 6.0 6.1 6.2 6.1 C. unicolor 301 77 134 69 185 5.0 5.2 5.3 5.8 C. unicolor 302 172 840 1260 7644 5.8 5.8 6.0 6.0 C. unicolor 303 76 151 133 407 5.3 5.8 5.4 5.8 C. unicolor 305 1025 420 176 210 5.5 5.5 5.5 5.7 C. gallica 142 105 332 470 504 5.3 5.7 5.6 5.7 M. tremelosus 206 605 504 181 66 4.9 4.3 4.5 4.5 P. tuberculosus 121 4 20 0 17 5.8 6.2 6.1 6.1 P. tuberculosus 131 0 0 2 8 6.0 6.2 6.0 6.0 C. striatus 978 4 0 0 20 6.1 6.1 6.1 5.8 MnP, U 1⁻¹ (610 nm) MnP, U 1⁻¹ (270 nm) Cultivation days 5 7 9 12 5 7 9 12 C. unicolor 300 437 79 67 42 645 17 0 0 C. unicolor 301 156 221 552 81 206 482 507 155 C. unicolor 302 34 40 41 70 52 43 26 0 C. unicolor 303 394 734 874 55 507 1015 576 95 C. unicolor 305 206 225 101 49 284 507 215 146 C. gallica 142 29 59 22 3 26 17 34 30 M. tremelosus 206 4 0 0 0 0 0 9 0 P. tuberculosus 121 22 56 152 160 0 0 0 0 P. tuberculosus 131 119 201 308 128 0 0 0 0 C. striatus 978 44 5 0 0 0 0 0 0 LiP, U 1⁻¹ Cultivation days 5 7 9 12 C. unicolor 300 1 2 3 0 C. unicolor 301 0 2 2 15 C. unicolor 302 4 3 2 0 C. unicolor 303 5 18 0 9 C. unicolor 305 1 4 1 30 C. gallica 142 9 12 12 3 M. tremelosus 206 0 0 0 0 P. tuberculosus 121 0 0 0 0 P. tuberculosus 131 0 0 0 0 C. striatus 978 0 0 4 0 Composition of medium, (g/l): KH₂PO₄—1.0; MgSO₄—0.5; CaCl₂—0.1; FeSO₄×7H₂O—0.005; peptone—1.0; yeast extract—2.0; veratryl alcohol—0.3; glycerol—10.0; MP—20.0. pH 5.0.

Laccase, U 1⁻¹ pH Cultivation days 5 7 9 12 5 7 9 12 C. unicolor 300 7392 6888 3654 9576 5.5 5.6 5.6 5.8 C. unicolor 301 4508 6552 2394 4620 4.7 4.8 5.0 5.0 C. unicolor 302 4620 7560 5516 12432 4.6 5.7 5.8 6.0 C. unicolor 303 2520 4340 2520 2016 4.3 5.0 5.0 5.2 C. unicolor 305 4620 5992 3276 5460 4.9 4.9 5.0 5.0 C. gallica 142 3528 2898 5292 2688 4.8 5.7 5.2 5.2 M. tremelosus 206 2982 3318 3570 3864 4.4 4.5 4.5 4.5 P. tuberculosus 121 13 42 34 121 4.7 5.6 5.5 5.7 P. tuberculosus 131 10 8 7 17 4.4 5.3 5,5 5.5 C. striatus 978 500 622 672 1596 4.7 4.6 4.6 4.6 MnP, U 1⁻¹ (610 nm) MnP, U 1⁻¹ (270 nm) Cultivation days 5 7 9 12 5 7 9 12 C. unicolor 300 423 92 30 14 980 120 34 0 C. unicolor 301 953 1122 760 808 2242 2761 705 1152 C. unicolor 302 16 24 13 24 0 17 9 0 C. unicolor 303 962 1072 313 22 2219 1376 237 56 C. unicolor 305 843 935 911 513 1213 1084 714 731 C. gallica 142 74 57 108 111 52 69 52 60 M. tremelosus 206 11 25 58 0 0 17 34 0 P. tuberculosus 121 701 898 591 124 0 22 0 0 P. tuberculosus 131 846 363 400 347 26 26 86 26 C. striatus 978 0 0 4 0 17 0 38 0 LiP, U 1⁻¹ Cultivation days 5 7 9 12 C. unicolor 300 47 98 196 0 C. unicolor 301 1 37 67 110 C. unicolor 302 6 74 103 0 C. unicolor 303 24 35 59 5 C. unicolor 305 47 73 25 70 C. gallica 142 3 121 107 6 M. tremelosus 206 0 0 0 0 P. tuberculosus 121 0 0 0 0 P. tuberculosus 131 47 0 0 2 C. striatus 978 7 0 0 0

2. Alginate Encapsulation of Enzyme Cocktail

Mycorernedation via Encapsulation and Controlled Release of Ligninolytic Enzymes from Alginate microparticles

The goal of this work is to develop the use of alginate encapsulation approaches for ligninolytic enzymes for the stabilization and controlled release in soils contaminated with target hydrocarbons. The ideal result will be the identification of the materials and methods yielding alginate microparticles meeting the following:

Small enough size that they can be dispersed in aqueous medium and sprayed onto soil

High active until loading (Units/mass of dispersion)

Long term stability

Demonstrated ability to degrade hydrocarbons in contaminated soil.

Task 1—Investigate the Effect of 1VIn2+ on Enzyme Activity in Alginate Beads

We will test the effect of the inclusion of Mn2+ on the encapsulation of three ligninolytic enzymes: lignin peroxidase (LiP), manganese-dependent peroxidase (MnP) and laccase. We will evaluate the capsules' size and enzyme loading. A promising formulation will be selected for investigation into methods to reduce the size.

Task 2—Investigate Methods of Reducing the Alginate Bead Size

Methods to be investigated are microemulsion and extrusion techniques. These methods will be evaluated based on particle size, dispersability, and potential sprayability. In addition, we will evaluate the enzyme loading in each microcapsule (units/mass capsule), The most promising method(s) will be chosen for testing long term stability and efficacy in contaminated soil.

Task 3—Stability and Efficacy

One or more promising methods will be chosen for final stability and efficacy tests. These will be tested against unencapsulated control enzymes. Results will be gauged on both the ability of the capsule to improve enzyme stability as well as ability to degrade the target hydrocarbons in contaminated soil.

TABLE 2 Experimental Test Matrix for Tasks 1 and 2. Alginate concentration will be constant at 20 mg/mL (2% w/v) based on prior results and published data. Enzyme loading will be chosen based on desired active units per mass of alginate. Processing Variables Compositional Variables Cross Method For Link Density/Amendment Extrusion, Excursion For Emulsion Run C_(BaCl2) C_(CaCl2) C_(MnCl2) Spray, Flow Rate Stir Rate C_(tween80) Task (#) (mM) (mM) (mg/mL) Emulsion (mL/hr) (RPM) (mg/mL) 1 1 0 10 100 Extrusion 10 2 1 0 100 Extrusion 10 2 3 TBD TBD 100 Extrusion 200 2 4 TBD TBD 100 Extrusion 400 2 41 TBD TBD 100 Extrusion 200 4

Encapsulation Experiments Continued

Background/Executive Summary

In the first round of alginate experiments, we saw that the conditions were not able to yield discrete alginate particles. The beads did not solidify and most of the collection bath became brown indicating that enzyme was not efficiently encapsulated. We hypothesized that the concentration of the crosslinking divalent ions were too low, and that this was resulting a weak encapsulating hydrogel matrix. To test this, we amended our test matrix to test three combinations of crosslinking divalent ions, each with higher concentrations of CaCl2 and BaCl2. The concentration of MnCl2 was kept constant at 100 mM since this is already high, and because the Mn2+ ion place a role in the enzyme activity in addition to crosslinking the alginate. The result confirmed our hypothesis and increasing the CaCl2 and BaCl2 concentrations yielded much more robust, and spherical beads. However, the collection bath still showed some brown color. We will run enzyme activity tests to quantify the units per bead. This will be done by dissolving a bead in 55 mM sodium citrate and running an assay on the solution. We will also run assays on the collection bath solutions.

Approach Materials

A stock solution of Alginate in DI water was prepared at 40 mg/mL and dissolved by heating in an autoclave. Other stock solutions were prepared accordingly. ABTS (10 mg/mL), CaCl2 (200 mM), MnCl2 (200 mM) and BaCl2 (10 mM) in DI water. Enzyme (MnP from C, unicolor 300) was used as received. This was a vicious dark brown liquid with the following estimated enzyme concentrations: laccase (437 U/mL), MnPA270 (265 U/mL), yielding a total enzyme concentration of 840 U/mL.

Procedure

-   -   1. To a 20 mL glass scintillation vial, add:         -   a. 3.3 mL Alginate Stock (40 mg/mL; via 10 mL B-D syringe             and 18 gauge hypodermic         -   b. 2.9 mL enzyme (MnP from C unicolor 300; via 10 mL B-D             syringe and 18 gauge hypodermic         -   c. 0.132 mL of ABTS (10 mg/mL in DI water; via volumetric             pipette)         -   d. 0.289 mL of DI water (via volumetric pipette)     -   2. This resulted in pre-alginate solution with the following         concentrations:         -   a. 365 U/mL total enzyme (composed of the following enzymes)         -   b. 190 U/mL Laccase         -   c. 115 U/mL MnPA610         -   d. 60 U/mL MnPA610         -   e. 0.2 mg/mL ABTS         -   f. 20 mg/mL Alginate     -   3. This solution was dispensed into collection baths with         various concentrations of crosslinking ions (shown in Table 1         below), including MnCl2, CaCL2. For each run, 1 mL of         pre-alginate solution was dispensed (at 10 mL/hr) through a 22         gauge stainless steel, blunt tipped needle into 50 mL of         crosslinking solution in the collection bath. As the droplets         hit the solution, they immediately solidified and sank to the         bottom of the dish. The dish was rotated by hand to avoid         accumulation of beads in one place in the dish. Note, that         throughout the dispensing step, the collection bath gradually         adopted a light brown color, indicating that some of the enzyme         was diffusing from the beads into the collection bath.     -   4. After 1 mL was dispensed, the dish was left to sit for at         least 30 minutes to allow the crosslinking to complete. Then the         liquid was pipetted off the stored as the decantate. The dry         beads were imaged using a camera (images were later analyzed for         particle diameter using ImageJ software). These were then         resuspended in 1 mL of DI water and stored in the refrigerator.

Results

The two of the runs (samples 3 and 4) yielded discrete beads that were able to be measured using ImageJ. If we assume that all of the enzyme in the pre-alginate solution was encapsulated in the beads (i.e. 100% encapsulation efficiency), and we estimate the bead volume from the measured diameters, we can estimate the enzyme concentration per bead (U/bead). This is shown in the Table below. Images and particle size distributions are shown in Figure below.

TABLE 1 Bead size and Estimated Enzyme loadings for successful runs (3 and 4). Bead Date Run C_(BaCl2) C_(CaCl2) C_(MnCl2) Diameter Enzyme Loading (U/bead) (y/m/d) (#) (mM) (mM) (mg/mL) (mm) Laccase MnP₂₇₀ MnP₆₁₀ 2015 Mar. 18 1 10 100 0 NA NA NA NA 2015 Mar. 18 2 0 100 1 NA NA NA NA 2015 Mar. 23 3 50 100 2.5 2.8 ± 0.1 2.18 1.32 0.690 2015 Mar. 23 4 100 100 0 3.0 ± 0.1 2.69 1.63 0.848 2015 Mar. 23 5 0 100 5 NA NA NA NA

It looks like sample 3 had better encapsulation efficiency than 4. Although it could be improved. Most of the enzyme is being lost in the bath. There are three things we could try to improve the encapsulation efficiency.

-   -   (1) Test higher concentrations of crosslinkers (CaCl2, and         BaCl2)     -   (2) Store the beads dry and then disperse them in water when         we're ready to test     -   (3) Reduce the amount of time that the beads are sitting in the         bath before collection

Note: Encapsulation efficiency is based on enzyme activity assays. Thus, it is possible that there is some de-activation enzyme encapsulated that was not detected by the assay. This would mean that the actual encapsulation efficiency of the enzyme was higher by some unknown amount, and that this was effect by enzyme de-activation in the process. 

1-24. (canceled)
 25. An enzyme formulation comprising a suite of enzymes comprising manganese peroxidase, lignin peroxidase, and laccase, ABTS, H₂O₂, and veratryl alcohol in a cross-linked hydrogel matrix.
 26. The formulation of claim 1 in the form of beads and wherein the hydrogel matrix comprises manganese.
 27. The formulation of claim 2 wherein the beads have a diameter within a range of from about 1.5 mm to about 5 mm.
 28. The formulation of claim 1 wherein the cross-linked hydrogel comprises calcium alginate, manganese alginate, zirconium alginate, calcium poly(aspartate), manganese poly(aspartate) or zirconium poly(aspartate).
 29. The formulation of claim 1 dispersed onto soil.
 30. A method of making an enzyme formulation, comprising: providing a solution comprising: a suite of enzymes comprising manganese peroxidase, lignin peroxidase, and laccase, a cross-linked hydrogel, ABTS, and veratryl alcohol; and adding the solution dropwise to a bath of crosslinkers.
 31. The method of claim 6 wherein the bath is a stirred bath comprising MnCl₂, CaCl₂ and BaCl₂.
 32. The method of claim 7 wherein the bath is a stirred bath comprising MnCl₂.
 33. A method of degrading a material, comprising: treating the material with the formulation of claim 1, wherein the material is selected from the group consisting of hydrocarbons, vulcanized rubber, synthetic rubber, natural rubber, vulcanized polymers and perfluorinated compounds.
 34. The method of claim 9 wherein the hydrocarbons include total petroleum hydrocarbons.
 35. The method of claim 9 wherein the hydrocarbons include polycyclic aromatic hydrocarbons.
 36. The method of claim 9 which includes remediating a soil contaminated with the material. 